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Negative mutation screening of the NOG, BMPR1B, GDF5, and FGF9 genes indicates further genetic heterogeneity of the facioaudiosymphalangism syndrome

van den Ende, Jenneke J.; Borra, Vere; Van Hul, Wim

doi: 10.1097/MCD.0b013e3283590986

We report on a patient with a clinical phenotype showing all the features of the multiple synostoses syndrome or the facioaudiosymphalangism syndrome, including symphalangism, condunction deafness, and the typical facies. Previously, it was shown that this condition is genetically heterogeneous with initially mutations described in the NOG gene, coding for Noggin, an extracellular antagonist of bone morphogenetic proteins. Noggin also interacts with growth differentiation factor 5 (GDF5), in which mutations have also been described in families with symphalangism. The latter is also the case for the BMP receptor BMPR1B to which GDF5 binds. Finally, a mutation in another growth factor, fibroblast growth factor 9, was found in a family with multiple synostoses syndrome. In our patient, we could, however, not show a causative mutation in any of these genes, providing evidence for further genetic heterogeneity of this syndrome.

Department of Medical Genetics, University Hospital Antwerp, University of Antwerp, Antwerp, Belgium

Jenneke J. van den Ende and Vere Borra contributed equally to the writing of this article.

Correspondence to Jenneke J. van den Ende, MD, Department of Medical Genetics, University Hospital Antwerp, University of Antwerp, Prins Boudewijnlaan 43, 2650 Antwerp, Belgium Tel: +32 3 275 9774; fax: +32 3 275 9722; e-mail:

Received August 1, 2011

Accepted August 7, 2012

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Proximal symphalangism has already been described as an autosomal dominant trait by Cushing (1916). Maroteaux et al. (1972) described a condition with proximal symphalangism of the fingers, carpal, and tarsal fusion among other skeletal abnormalities. Congenital conduction deafness, because of stapes fixation in the oval window, was present, in combination with a typical facies, showing a broad, hemicylindrical nose, with lack of alar flare, a narrow upper lip, strabism, and hypermetropia. He called the condition ‘multiple synostoses syndrome’, but later, other names were suggested, such as WL symphalangism (Herrmann, 1974) and facioaudiosymphalangism syndrome (FAS) (Hurvitz et al., 1985).

A gene involved in proximal symphalangism was localized on chromosome 17q21–22, Polymeropoulos et al. (1995) and Krakow et al. (1998) reported the linkage of the multiple synostoses syndrome (SYNS1) to the same locus. In 1999, mutations were identified in the NOG gene (MIM# 602991), encoding the polypeptide Noggin (Gong et al., 1999), in a family with dominantly inherited multiple synostoses syndrome, as well as in six unrelated families with proximal symphalangism, leading to the assumption that these are allelic disorders. Since then, NOG mutations have been described in several overlapping conditions with proximal symphalangism (Takahashi et al., 2001; Brown et al., 2002; Potti et al., 2011). However, in some patients with these conditions, no mutation in the NOG gene could be found, leading to the assumption that the syndrome is genetically heterogeneous.

Both Akarsu et al. (1999) and Dawson et al. (2006) have reported missense mutations in the growth differentiation factor 5 (GDF5) gene, also called cartilage-derived morphogenetic protein 1 (CDMP1) (MIM# 601146), in patients with multiple synostoses syndrome. Similarly, Seemann et al. (2005) showed a missense mutation in the GDF5 gene in a family with proximal symphalangism of the fifth proximal interphalangeal (PIP5) joints and the fifth distal interphalangeal (DIP5) joints and to a lesser extent in the fourth fingers. In this family, no other skeletal features were present. This was confirmed by the finding of Wang et al. (2006) of another missense mutation in GDF5 in two fifth-generation Chinese families with proximal symphalangism.

Lehmann et al. (2006) published the case of a German woman with hand abnormalities resembling Brachydactyly type C with features of proximal symphalangism, who showed a missense mutation in the BMPR1B gene (MIM# 603248), which is the receptor of the ligand GDF5.

Recently, a mutation in fibroblast growth factor 9 (FGF9, MIM# 600921) was reported by Wu et al. 2009 in a Chinese family with multiple synostoses syndrome (SYNS3).

In this report, we present a patient with a clinical diagnosis of FAS, but with no detectable mutation in the above-mentioned genes, providing evidence for further genetic heterogeneity of this syndrome. We will review the genes already described and discuss their role in the BMP/GDF pathway and the possible causative role in the symphalangism syndromes.

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Case report

The proband, a 15-year-old girl with nonconsanguineous Flemish parents, was referred to the genetics department about 10 years ago, because of a suspicion of a collagen disorder. At birth, she had an umbilical hernia. Her birth weight was 3.690 kg and birth length was 54 cm. As a child, she had a hemangioma at the point of her nose. She now presented with a thin, translucent skin, strabism, and hypermetropia. She had multiple joint problems, with recurrent pain in the knees, fifth fingers, and toes, but in contrast to hypermobility, she had reduced joint movement, especially of the fifth proximal interphalangeal (PIP) joints, and to a lesser extent of the first and fourth PIP joints. She could not make a fist.

Furthermore, she showed progressive hearing loss. Intelligence was normal.

Both parents, as well as a younger brother and sister were unaffected.

Physical examination showed a tall girl [1.75 m (90–97th centile], with a relatively small head [OFC 52 cm (2nd centile)]. She had large hands (97th centile) and feet (shoe size 45).

The face was slightly asymmetric, with upslant of the eyes, a hemicylindrical nose with a bulbous tip, where the bleached hemangioma could be seen (Fig. 1). There were slightly visible veins on the thorax. No scoliosis or flat feet could be found.

Fig. 1

Fig. 1

There was a lack of a flexion crease at the right fifth PIP joint and no flexion possible of the first and fifth PIP joints bilaterally. Broad thumbs were noticed (Fig. 2).

Fig. 2

Fig. 2

Radiographs showed a normal spine, fusion of the PIP joints of the fifth ray bilaterally, undertubulation of both second metacarpals, and short first metatarsal bones bilaterally, but no fusions or tarsal coalition (Fig. 3). A computed tomography-scan of the middle ear showed no visible abnormalities of the ossicles or otosclerosis. Hearing tests showed conductive hearing loss of about 20 dB, especially at low frequencies.

Fig. 3

Fig. 3

Additional investigations showed normal chromosomes.

Because of the lack of hyperlaxity, easy bruising, delayed wound healing, and luxations we ruled out a collagen disorder.

The combination of symphalangism and deafness, together with the typical facial appearance (hemicylindrical nose, upslant) and ocular problems, indicated a diagnosis of the facioaudiosymphalangism (FAS) syndrome or multiple synostoses syndrome. Because in patients with this syndrome the hearing loss is due to congenital fixation of the stapes, our patient underwent a stapedectomy that was successful.

The patient returned to our department 10 years later for preconceptual advice to know the recurrence risk for future children. A year before, she had suffered seizures twice, for which she was treated with lamotrigin. In the meantime, several genes had been shown to be involved in cases of multiple synostoses syndrome, enabling molecular analysis.

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Molecular genetics

Materials and methods

Mutation analysis

Peripheral blood was drawn from our patient. DNA was extracted from the blood samples using standard procedures.

The coding regions of the NOG, GDF5, BMPR1B, and FGF9 genes were sequenced.

All genes were amplified by a GoTaq DNA polymerase-mediated PCR (Promega Corporation, Madison, Wisconsin, USA). The PCR amplifications were carried out with a set of primers covering the exons and intron–exon boundaries.

Sequencing reactions were performed using the ABI BigDye Terminator v1.1 Cycle Sequencing Ready Reaction Kit (Applied Biosystems Inc., Foster City, California, USA). Purification with the BigDye XTerminator Purification Kit (Applied Biosystems Inc.) was carried out to remove unincorporated BigDye terminators, after which fragments were analyzed on an ABI 3130 Genetic Analyser (Applied Biosystems Inc.).

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Single-nucleotide polymorphism analysis

Single-nucleotide polymorphism (SNP) analysis was carried out to ensure that both copies of each gene were present. Intragenic SNPs were found in the NCBI SNP database ( Primers were developed to create amplicons containing several SNPs. These amplicons were PCR-amplified and sequenced as described above.

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The patient showed normal chromosomes by karyotyping. Mutation screening of the four genes implicated in these conditions (NOG, GDF5, BMPR1B, and FGF9) did not show a mutation in the patient. To ensure that both copies of the gene were present and to rule out large deletions, SNPs were analyzed. Heterozygous SNPs were found in all genes at several locations (Table 1), which indicated that both copies of each gene are present.

Table 1

Table 1

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Several names are used for overlapping conditions in which proximal symphalangism is the common feature. In our patient, the best-fitting diagnosis would be the FAS as she showed the typical face, with a hemicylindrical nose, upslanting palpebral fissures, and strabism. She had symphalangism of PIP5 bilaterally. She showed progressive hearing loss, and, although on computed tomography-scan no stapes fixation could be visualized, stapedectomy led to considerable improvement in hearing. She has a relatively tall stature, but with disproportional large feet. She developed seizures, which have not been described before as part of the syndrome. The presence of broad thumbs, hyperopia, and lack of fusions in carpals or tarsals also fit the diagnosis of ‘stapes ankylosis with broad thumbs and toes’ (SABTT) (Teunissen and Cremers, 1990; Potti et al., 2011). These conditions are probably allelic and belong to the same ‘symphalangism spectrum disorder’.

The multiple synostoses syndromes were found to be genetically heterogeneous, with the currently four known genes involved being NOG (SYNS1), GDF5 (SYNS2), FGF9 (SYNS3), and BMPR1B (BDC/SYM1-like phenotype).

Table 2 presents an overview of the mutations in each gene causing these conditions.

Table 2

Table 2

The fact that mutations in the gene encoding Noggin underlie a synostoses phenotype correlates with its function. Noggin is known to bind and inactivate Bone Morphogenetic protein (BMP)-4, belonging to the transcription growth factor-β (TGF-β) superfamily. In addition to binding to BMP4, Noggin also binds to several other BMP family members, all involved in early growth and differentiation. BMP4 is expressed at multiple sites, including developing bones, and is necessary for joint formation (Brunet et al., 1998). It binds to specific receptors on the surface of cells and then transduces signals that activate particular genes, leading to the transformation of mesenchymal cells into cartilage-forming and bone-forming cells (Balemans and Van Hul, 2002).

The activity of the BMPs is controlled at many levels. Outside the cell, proteins such as Noggin, Chordin, and Follistatin bind BMPs and inhibit their binding to cell surface receptors. Inside, the cell activity is controlled by signal-transducing and inhibitory Smad proteins. In addition, a number of negative regulators of BMP action exist within the nucleus (Ebara and Nakayama, 2002).

The defect in FAS probably occurs in the eighth week of gestation, when interzones of the finger joints differentiate, before the formation of the joint cavity. Subsequently, blood vessels grow into the adjacent connective tissue, resulting in the formation of synovial cavities that separate the cartilaginous surfaces of the bones. This might be the critical stage in which mutations in the NOG gene lead to defective joints that fuse later in life.

In mice lacking Noggin, cartilage condensations initiated normally but developed hyperplasia and initiation of joint formation failed. Excess BMP activity in the absence of Noggin antagonism may enhance the recruitment of cells into cartilage, resulting in oversized growth plates (Brunet et al., 1998).

In humans, heterozygous mutations cause a reduction in the secretion of functional dimeric Noggin, and lead to several syndromes with symphalangism and/or stapes fixation.

A functional explanation for the finding of mutations in GDF5 and BMPR1B can be hypothesized. GDF5 is also a member of the TGF-β superfamily. It is expressed at sites where joints will form, between skeletal elements in developing mouse limbs (Storm and Kingsley, 1999). GDF5 is processed from a larger precursor protein and forms homo–heterodimers with other BMPs. It binds to BMP receptors (BMPR1 and 2), leading to the activation of the Smad-dependent pathway, which regulates the transcription of specific target genes that are involved in bone formation. In the absence of Noggin, there will be dysregulation of BMP activity and consequently failure to activate GDF5 necessary to initiate joint development.

All genes mentioned in Table 2 were analyzed in our patient, but no mutations were found. As most disease-causing mutations occur in the coding regions of genes, we focused on the exons and the intron–exon boundaries for mutation analysis. Nevertheless, it is possible that mutations could occur in the promoter or the enhancer regions and affect gene expression.

To ensure that both copies of each gene are present, SNP analysis was carried out. Heterozygous SNPs were found in all genes, implying that both copies are present. In this way, large deletions are ruled out. However, we cannot be absolutely sure that there are no small deletions in introns affecting splice sites or promoter regions.

Until now, only mutations in the NOG gene seemed to lead to the complete picture of FAS with symphalangism, deafness, and facial features, whereas the other genes did not show the specific face. However, our negative molecular data in this patient clearly indicate that there might be further genetic heterogeneity underlying multiple synostoses syndrome with the involvement of at least one additional gene. Considering the complexity of the BMP signaling pathway, further research is required to explain the unresolved cases of symphalangism with or without other features.

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V.B. is a holder of a Ph.D. grant of the Agency for Innovation by Science and Technology (IWT).

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Conflicts of interest

There are no conflicts of interest.

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                            BMPR1B; facioaudiosymphalangism syndrome; FGF9; GDF5; multiple synostoses syndrome; NOG

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